Both samples were measured qualitatively handheld UV lamp and quantitatively spectrofluorometer. Then, an elution buffer 10mM Tris, pH 8.
Lastly, a Western Blot was performed with binding of primary and secondary antibodies to prove that the protein of interest, rGFP, had indeed been expressed and purified. Prendergast, and Milton J. Figure Captions Figure 1.
The same procedure was done for the W and E samples by taking 50ul of each sample and adding 1 ml of the Bradford reagent. The gel was then removed, stained with Coomassie Blue, and de-stained so that the size and purity of each sample containing the rGFP protein could be determined.
Then, breaking buffer was pipetted into the column in 0. The graph showed the washes to have larger amounts of protein, as was expected since all the undesired proteins were being rinsed out. Inside the rGFPuv, is the chromophore structure that causes the fluorescence. GFP was found to be a protein of amino acids and had a molecular weight of 26, kDa.
By plotting the data [Absorbance nm vs BSA ug ], the standard curve was created. The gel was placed in a gel electrophoresis tank and filled with electrophoresis buffer ml H2O, 50ml 10X stock solution 30g Tris, g glycine, 10g SDSD per liter.
The same washing step was repeated 3 times before probing the membrane with 7ml of Sheep IgG anti-mouse IgG conjugated horseradish peroxidase polyclonal anti-serum solution and incubated.
The attempt to express and purify rGFP from these series of experiments was successful. The Western Blot also showed that the smaller bands were merely contaminants since they did not show up in the final product. The schematic diagram of rGFP from the E. The column was then washed with breaking buffer in order to take out any ethanol that the agarose had been stored in.
Because the antibodies were able to bind and the visible markings showed up in the Western Blot, this was able to prove that the bands on the gel were indeed rGFP.
Western blot of the rGFP samples. Once secured onto a ring stand with a clamp, a luer-lock was fastened onto the end of the syringe. It was located around the 34 kDa marker on the ladder, which is fairly close to the calculated MW of the rGFP protein.
Other bands smaller than this were also visible, but at this time could not be identified as a contaminant or rGFP with C-terminus degradation. This diagram displays the entire sequebce of the rGFP protein, coming out to be amino acids long.
The third elution E3 ended up having the most amount of rGFP and this was proven with the Bradford assay. After a final wash, 7ml of TMB substrate solution was added, and the membrane was incubated until the desired color intensity was achieved.
Once it polymerized, a comb was inserted to create the wells in which the samples would be placed. Once the stacking gel was polymerized, the gel was ready to be loaded with fractions of the E. Looking at figure 2, a closer look at the rGFP gene was analyzed.
Expression and Purification of recombinant Green Fluorescent Protein. Print Reference this. The Western Blot test, utilizing a nitrocellulous membrane, confirmed the expression of rGFP. The Western Blot confirmed that the correct bands were analyzed in the SDS-PAGE gel which E3 had an estimated purity ofindicating a yield of.
The purpose of these series of experiments was to express and purify recombinant Green Fluorescent Protein (rGFP) from the E. coli strain, BL21(DE3) by beginning with its purification via a Ni2+-agarose affinity chromatography column. SDS-PAGE gel, which was used to analyze our purification of rGFP from a crude extract, and the corresponding molecular weight.
This gel was made with a 12% resolving gel and a 5% stacking gel. Wildtype rGFP has a molecular weight for 27kDa. Lab 3: Expression of rGFP in E. coli. STUDY. PLAY. Shimomura --used to induce expression of rGFP.
G0. G culture before induction.
G3. E. coli is commonly used for protein expression and purification; we can easily make informed conclusions when using it for gene transformations.
2) The expression and purification of rGFP started with the growth of the bacterial culture and ended with elution of rGFP off the Ni+2 agarose coloumn. Describe one change to this process that you predict would result in an increase of the total yield of rGFP.
Any help would be appreciated. Thanks. 9) The expression and purification of rGFP started with the growth of the bacterial culture and ended with the elution of rGFP off the Ni+2-agarose column. Describe one change to this process that you predict would result in an increase of the total yield of rGFP%(14).Expression and purification of rgfp from